Double-kinetic fluorescence stopped-flow spectroscopy will be combined with spectroscopy-targeted site directed mutagenesis and chemical manipulation in order to experimentally examine protein folding pathways. Fluorescence experiments are performed on an instrument capable of simultaneous detection of five independent data axes in real-time during protein folding reactions (picosecond/nanosecond, millisecond/second, parallel intensity, perpendicular intensity, multiple emission wavelengths). High signal-to-noise time-resolved anisotropies and fluorescence lifetimes obtained every millisecond are utilized to directly measure fundamental properties of intermediate folded states of proteins. Experimental efforts are concentrated on the examination of the folding of yeast phosphoglycerate kinase (PGK) and E. coli alpha- subunit of tryptophan synthase (alpha-TS.) The following questions are being directly examined: 1) What is the exact time-scale upon which the picosecond/nanosecond local motions of tryptophans in the unfolded state become "coupled" to the global rotation of the native state during a folding reaction. 2) Do all domains (or subdomains) within a protein restrict this tryptophan motion with identical kinetics? 3) What is the hydrodynamic radius of initially collapsed protein states, and transiently populated "overexpanded" states? 4) Do tryptophans "pack- into" this initially collapsed structure? 5) What is the time-scale for the unfolding of native-like distances in PGK between: both ends of an alpha-helix?, adjacent alpha-helices?, the extreme ends of a single domain?, across a hinge between two domains? Using both multi-site single tryptophan stopped-flow anisotropy and multi-site stopped-flow energy- transfer measurements, millisecond" structural-and-dynamic" motion- pictures of protein folding will be determined. Real-time measurements of rotational dynamics, intramolecular distances, and solvent accessibility are emphasized. The design of a new "double-kinetic" pulse-height-analyisis analog-to-digital-converter is described, which combines parallel ADC's with multi-dimensional indexable histogramming memory arrays, to increase the inherent timing resolution of the double- kinetic method by another factor of 10X.